Expressing genes of interest in cells or tissues for therapeutic purposes often entails driving transcription with strong viral or constitutive promoters. However, such methods can lead to some undesired consequences. For example, tumorigenic transformation can occur in the transfected target cells from unregulated expression of potentially oncogenic cell conversion factors (1) or insertional mutagenesis by retroviral vectors (2, 3).
One solution is to incorporate some synthetic regulatory elements into the therapeutic constructs and this method has confers some control (4). However, this method also increases the system complexity and may require pharmaceutical agents to modulate promoter activity.
Another method is to assimilate appropriate regulatory elements to retain innate responsiveness of the engineered genes. Since transcription is a fundamental mechanism for controlling gene expression, incorporating naturally occurring regulatory elements can be useful in increasing fidelity and safety of engineered genes. For most eukaryotic genes, the majority of the information necessary for recapitulating developmental and spatial expression resides in the proximal promoter which can be a few hundred base-pairs (bp) long. However, endogenous transcriptional activity is difficult to achieve in engineered vectors with promoter fragments lacking their genomic context and distal elements. For instance, expression of genes linked to the β-globin promoter can achieve levels comparable to the endogenous promotor but requires inclusion of the β-globin locus control region, an extended enhancer located at an ectopic chromatin site (7, 8). Other promoters rely on similar compact distal enhancers (9) but locus control regions are unique and gene specific.
Several other strategies have been employed to enhance transcriptional activities of native promoters while retaining (or engineering) innate regulation: use of larger promoters (10), duplicating positive response elements (RE) (11) or more complex promoter fragments (12,13), fusing distal (14) or ectopic enhancers (15), and binary amplification systems (16). Appropriate targeting and responsiveness is theoretically possible by combining cis elements into “designer promoters” (17). For single response elements, highly inducible promoters have been constructed from a minimal TATA box by fusing multiple binding sites for hypoxia-inducible factor (HIF) binding sites with optimal spacing of orientation to achieve hypoxia responsiveness (18) or 7 T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) binding sites to achieve a β-catenin responsiveness (19).
However, attempts to achieve endogenous transcriptional activities from native mammalian promoters have not been as successful. New or significantly improved approaches are necessary to make native or synthetic promoters widely useful for genetic engineering in humans and other mammals.